The trace metal copper is essential for all life forms as a co-factor for many critical cellular enzymes such as cytochrome oxidase, Cu,Zn superoxide dismutase, dopamine beta hydroxylase and ceruloplasmin. However, due to its ability to engage in reactions generating reactive oxygen species that damage nucleic acids, proteins and membranes, Cu is also highly toxic. The importance of maintaining Cu balance is underscored by the existence of severe genetic diseases of Cu homeostasis and by the demonstration that Cu binding proteins are involved in diseases as Lou Gehrig's disease and prion diseases, and implicated in cancer and aging. This proposal describes avenues of investigation, using yeast as a model system, aimed at understanding the molecular mechanisms whereby cells appropriately acquire Cu, regulate the Cu transport process and regulate gene expression in response to changes in cellular Cu status. First, experiments are described to elucidate the topological arrangement of high affinity Cu transporters, localized to the plasma membrane, and to determine the mechanisms whereby these molecules specifically capture extracellular Cu ions and move these redox active metals across the plasma membrane in a highly controlled fashion. Second, experiments are described to understand how yeast cells fine tune the levels of the Ctr1 Cu transporter at the plasma membrane, as a function of environmental cu levels, by signaling events that lead to Cu induced endocytosis. Third, the function and mechanism of action of a novel Cu transport protein in facilitating the assembly of an active Cu transport complex at the plasma membrane will be determined. Fourth, the mechanisms whereby a Cu-sensing transcription factor couples intracellular Cu and iron levels by regulating expression of iron transport genes will be determined. The studies described in this proposal will provide fundamentally important information on the mechanisms whereby all cells maintain normal Cu homeostasis.